Fig. 3

Transgene Expression from the strong heterologous promoter is not entirely locus-dependent. CRISPR-mediated integration of CMV-driven EGFP and promoter-less DsRed2 in the predicted GSH loci and non-GSH locus was performed in DF1 cell lines. A Schematic depiction of CMV-EGFP-expressing heterogeneous cell pools at the end of MTH2. (B-a, C-a, D-a) CRISPR-mediated integration of DsRed2-CMV-EGFP in cROSA, cHIPP, and cOVA loci. (B-b, B-c, C-b, C–c, D-b, D-c) Light and fluorescence microscope images of the cells expressing EGFP heterogeneously driven by the CMV promoter (Scale bar: 100μm). (B-d, C-d, D-d) Flow cytometry results from EGFP-expressing cROSA, cHIPP, and cOVA cells (each in triplicate). Non-transfected cells were used as the negative control. No expression signal was detected in the red channel. E Mean fluorescent intensity (MFI) index of the cOVA group was higher than that in the cHIPP and cROSA groups. F The integrated density (ID) index of the cOVA group was higher than that in the cHIPP and cROSA groups. G The copy number (CN) of EGFP transcripts in the cOVA group was higher than that in the cHIPP and cROSA groups. **: p < 0.05, ***: p < 0.005, and ****: p < 0.0001 are statistically significant. Avg.: The average expression of EGFP. Exp: Experiment. N: Number